Mayer hematoxylin

Males were sacrificed by cervical dislocation. Testis and epididymis were fixed in 4% paraformaldehyde in PBS and were processed for plastic sectioning using a Technovit 8100 instrument (Kulzer, Wehrheim, Germany) according to the manufacturer’s instruction. Briefly, fixed testes were washed in PBS at 4 °C for 1 h, dehydrated in acetone at 4 °C for 1 h, infiltrated with in mixed solution of Technovit 8100 basic solution and hardener 1 (1.5 mL of basic solution plus 9 mg of hardener 1 per sample) at 4 °C for 2–6 h, and then embedded after adding 50 μl of hardener 2. For analysis of mouse testes, 5 μm sections were treated with 1% periodic acid for 10 min, followed by treatment with Schiff reagent (#193-08445, FUJIFILM Wako Pure Chemical, Osaka, Japan) for 20 min. The sections were stained with Mayer hematoxylin (#131-09665, FUJIFILM Wako Pure Chemical) solution prior to imaging and observed under microscope. For analysis of mouse epididymis, 5 μm sections were treated with Mayer hematoxylin solution for 3 min, then were stained with Eosin solution (1% Eosin Y solution [#051-06515, FUJIFILM Wako Pure Chemical] mixed with 80% ethanol in 1:2 ratio), and then added 0.5% of total volume of acetic acid for 2 min. Stained sections were then observed under microscope.

The prepared tissue sections were deparaffinized by consecutive treatment with xylene and different concentrations of alcohol for 5 min for each immersion. Mayer hematoxylin (Wako, Japan) was used for nuclear counterstaining, and counterstaining was conducted by immersing each section in an eosin solution (1% eosin). The sections were dehydrated and hyalinized in alcohol before visualization with a microscope (Olympus Tokyo, Japan).